Circulating platelets are activated by a variety of endogenous substances, e.g., thrombin, ADP, but most particularly be altered or damaged blood vessel surfaces. Although the exact intraplatelet calcium ion concentration [Ca++] is not known, it is clear from threshold [Ca++] needed to activate intraplatelet systems that the unactivated platelet must maintain very low [Ca++] i.e., 0.1 MuM or less. Failure to maintain low [Ca++] must therefore lead to inappropriate platelet activation and increased susceptibility to thrombosis. In view of the large (1,000 fold) concentration gradient and the electrical gradient (-60 mV) for Ca++, low [Ca++] can be maintained by impermeability, by sequestration into storage sites and by extrusion. It is clear that platelets are not impermeable to Ca++ therefore extrusion against the Ca++ electrochemical gradient is vital. Ca++ extrusion can occur in process similar to Ca++ sequestation, i.e., a Ca++ -stimulated ATP-requiring reaction supporting Ca++ movement across a membrane and/or in exchange for an ion or ions moving into the platelet. In other cells, e.g., heart, intracellular Ca++ is extruded in exchange for extracellular Na+(Na+). A Na+ gradient (Nao+)/(N2ai+) is maintained by a [Nai+]-stimulated ATP-requiring reaction supporting Nai+ extrusion; thus Cai extrusion may be indirectly supported by the Na efflux pump. The Ca2i++/Nao+ inlux exchange (or the reverse) appears to be mediated by a voltage-sensitive carrier within the membrane that simultaneously binds and transports Na+ and Ca++. The specific aim of this proposal is to study the platelet Cai++ extrusion mechanism(s). ATP-dependent-Ca++ transport into vesicles formed from plasma (surface) membrane fragments or from intraplatelet membrane fragments, will be compared with respect to kinetics of Ca++ uptkae, inhibitor specificity and susceptibility to modulator proteins, e.g., calmodulin. The plasma vesicle Nao+/Cai++ and Cao++/Cai++ exchange characteristics (in the absence of ATP) will be studied. Finally effort will be directed toward assessing the relative contribution of the two extrusion mechanisms to Ca++ extrusion from the intact platelet.